Triggerable Protocell Capture in Nanoparticle-Caged Coacervate Microdroplets

Controlling the dynamics of mixed communities of cell-like entities (protocells) provides a step toward the development of higher-order cytomimetic behaviors in artificial cell consortia. In this paper, we develop a caged protocell model with a molecularly crowded coacervate interior surrounded by a non-cross-linked gold (Au)/poly(ethylene glycol) (PEG) nanoparticle-jammed stimuli-responsive membrane. The jammed membrane is unlocked by either exogenous light-mediated Au/PEG dissociation at the Au surface or endogenous enzyme-mediated cleavage of a ketal linkage on the PEG backbone. The membrane assembly/disassembly process is used for the controlled and selective uptake of guest protocells into the caged coacervate microdroplets as a path toward an all-water model of triggerable transmembrane uptake in synthetic protocell communities. Active capture of the guest protocells stems from the high sequestration potential of the coacervate interior such that tailoring the surface properties of the guest protocells provides a rudimentary system of protocell sorting. Our results highlight the potential for programming surface-contact interactions between artificial membrane-bounded compartments and could have implications for the development of protocell networks, storage and delivery microsystems, and microreactor technologies.


Preparation and characterization of Au nanoparticles
Au nanoparticles were synthesized by the following protocol. 100 μL HAuCl 4 aqueous solution (100 mg/mL) was added to 80 mL deionized water and heated to 60 °C. 0.049 g sodium citrate, 0.01 g tannic acid and 0.0036 g K 2 CO 3 were quickly added into the aqueous solution and the aqueous mixture was allowed to react under magnetic stirring (400 rpm) at 60 °C for 10 min to afford a Au-nanoparticle dispersion. The reaction was undertaken in a 250 mL roundbottom flask, which was cleaned with freshly prepared aqua regia before usage. The prepared Au-nanoparticle suspension was used without further purification.
Transmission electron microscopy (TEM) was used to characterize the morphology and diameter of the as-synthesized Au nanoparticles. UV-vis spectroscopy was used to determine the concentration of the as-synthesized Au nanoparticles. A commercial Au-nanoparticle suspension (Sigma-Aldrich, product number 741949, concentration 5.5 ± 0.5 x 10 13 particles/mL) was used as a standard solution. The maximum absorption values between 500 and 550 nm of the home-synthesized and commercial Au-nanoparticle solutions were set to 0.712 (A 510 ) and 0.715 (A 523 , diluted to 69% of the original concentration), respectively, so that the concentration of the as-synthesized Au-nanoparticle solution used was ca. 3.8 x 10 13 particles/mL.
(2) Synthesis of the HOOC-PEG Poly(ethylene glycol) (0.1 mmol), DMAP (0.06 mmol) and succinic anhydride (0.4 mmol) were dissolved into 50 mL chloroform in a round-bottomed flask with a magnetic stirrer and reflux condenser. A stream of argon was bubbled through the solution for 15 minutes and the reaction vessel was heated to 60 °C. The reaction was stirred at 60 °C for 18 hours. The solvent was removed by rotary evaporation. The crude product was dissolved in 10 mL dichloromethane and then precipitated by dropwise addition into 200 mL cool diethyl ether. HOOC-PEG was collected by filtration. 1 H-NMR (400 MHz, CDCl 3 ): δ(p.p.m.) 4.23 (t, 4H), 3.7-3.5 (m, H of PEG main chain), 2.61 (m, 8H).

(4) Synthesis of the TsO-PEG
Poly(ethylene glycol) (1 mmol) and triethylamine (10 mmol) were dissolved in 70 mL dichloromethane in a round-bottomed flask with magnetic stirrer and the reaction vessel was cooled to 0 °C. 4-Toluenesulfonyl chloride (10 mM) was added into the solution. The reaction mixture was left at room temperature with constant stirring for 24 hours. Then 100 mL deionized water was added and stirred for 1 hour. The organic layer was rotary evaporated and followed by dissolving with 40 mL dichloromethane and then precipitated by dropwise addition into 400 mL cool diethyl ether.

(5) Synthesis of the N 3 -PEG
TsO-PEG (0.5 mmol) and sodium azide (5 mmol) were dissolved into 10 mL DMF in a round-bottomed flask with magnetic stirrer and reflux condenser. The reaction vessel was heated to 95 °C and stirred at 95 °C for 18 hours. After cooling down to room temperature and filtration, DMF was evaporated under vacuum. The crude product was dissolved in 10 mL dichloromethane and washed twice with 20 mL saturated NaCl aqueous solution. The dichloromethane layer was dried over sodium sulfate, and precipitated by dropwise addition into 200 mL cool diethyl ether. The product was collected by filtration. 1 H-NMR (400 MHz, CDCl 3 ): δ(p.p.m.) 3.7-3.5 (m, H of PEG main chain), 3.37 (t, 4H).
(6) Synthesis of the NH 2 -PEG N 3 -PEG (0.5 mmol) and triphenylphosphine (2.5 mmol) were dissolved into 50 mL methanol in a round-bottomed flask with magnetic stirrer and reflux condenser. The reaction vessel was heated to 65 °C and stirred at 65 °C for 12 hours. After cooling down to room temperature and filtration, methanol was evaporated under vacuum. The crude product was dissolved into 20 mL dichloromethane and added dropwise into 200 mL cool diethyl ether. The product was collected by filtration. 1 H-NMR (400 MHz, CDCl 3 ): δ(p.p.m.) 3.7-3.5 (m, H of PEG main chain), 2.81 (m, 4H).

(7) Synthesis of the RITC-PEG-NH 2
NH 2 -PEG (1 g) was dissolved into 10 mL deionized water in a 25 mL glass vial. 10 mL carbonate buffer (100 mM, pH=9) was added into the vial followed by addition of RITC solution (5 mg dissolved into 2 mL DMSO). The mixture was stirred at room temperature for 12 hours, purified by dialysis (24 hours, 4 L water replaced three times) and then lyophilized to get product. The 1 H-NMR showed no difference with the 1 H-NMR of NH 2 -PEG because of the low grafting ratio of RITC.

(12) Synthesis of the RITC-PEG-DSC
RITC-PEG-NH 2 (1 g), triethylamine (100 μL) and disuccinimidyl carbonate (DSC, 100 mg) were dissolved in 8 mL dichloromethane in a round-bottomed flask with a magnetic stirrer. The reaction was stirred at room temperature and left to react for 18 hours. The dichloromethane solution was added into 400 mL diethyl ether dropwise. The product was collected by filtration and vacuum dried.

(13) Synthesis of the RITC-PEG-AE
RITC-PEG-DSC (1 g) triethylamine (30 μL) and 2,2-Bis(aminoethoxy)propane (33 μL) were dissolved in 6 mL dichloromethane in a round-bottomed flask with a magnetic stirrer. The reaction was stirred at room temperature and reacted for 24 hours. The dichloromethane solution was added into 300 mL diethyl ether dropwise. The product was collected by filtration and vacuum dried.

(14) Synthesis of the RITC-PEG-AE-TA
RITC-PEG-AE-TA was synthesized using the same procedure as for the synthesis of TA-PEG, with the exception that RITC-PEG-AE-TA was used instead of poly(ethylene glycol).

(15) Synthesis of the TA-PEG-ME
TA-PEG-ME was synthesized using the same procedure as for the synthesis of TA-PEG, with the exception that poly(ethylene glycol) methyl ether (PEG-ME) was used instead of poly(ethylene glycol) (PEG).

Transmission electron microscopy
A batch of fresh-prepared Au/TA-PEG-caged coacervate droplets was left to settle for 5 minutes. Then 10 μL solution was withdrawn from the top layer and diluted by addition of 90 μL supernatant solution. After gentle shaking, 5 μL of the diluted solution was carefully dropped onto a carbon film-loaded copper grid. The sample-loaded copper grid was left for 5 minutes followed by freeze drying to remove the water phase. The mounted sample was characterized using a FEI Tecnai 20 TEM (200 kV).

Preparation of fluorescently labelled caged coacervate droplets
RITC-labelled PEG derivatives (RITC-PEG-TA, Mw = 20k) and FITC-labelled CM-dex (FITC-CMdex) were used to determine the localization of the coacervate components by confocal laser scanning microscopy (CLSM). A coacervate dispersion was prepared by mixing 400 μL PDDA (400 mM), 900 μL CM-dex (370 mM) and 10 μL FITC-CM-dex (2 mg/mL). 10 μL of the coacervate suspension was then mixed with 100 μL Au-nanoparticle solution. 2 μL aqueous RITC-PEG-TA (10 mg/mL) was added into the solution under vigorous mixing and the dispersion of membranized coacervate microdroplets left unstirred to allow sedimentation. The upper layer of the solution containing unreacted RITC-PEG-TA was then replaced by the same volume of native coacervate supernatant.

Influence of MTA-PEG and Au concentrations on membrane stability
The stability of the membranized coacervate droplets was determined by the degree of coalescence, which was assessed by monitoring the size of the caged coacervate microdroplets using optical microscopy. 10 μL of the coacervate droplets (prepared by mixing 900 μL CM-dex and 250 μL PDDA) was mixed with a Au nanoparticle solution and MTA-PEG (10 mg/mL) aqueous solution. The final concentration of Au nanoparticles varied from 1.38 x 10 13 to 7.59 x 10 13 per mL. The final concentration of MTA-PEG varied from 1.8 to 45 μM. If the diameter of the largest membranized coacervate droplets was above 200 μm, the samples were regarded as highly unstable.

Reversibility of Au/TA-PEG membrane assembly
60 μL of a freshly prepared aqueous dispersion of the membranized coacervate droplets (stabilized by Au/TA-PEG) were injected into a sample holder. The droplets were illuminated at 290-390 nm for 5 min from the top at a distance of ca. 1 cm. The aqueous mixture was then withdrawn, added to a centrifuge tube, and vigorously sheared for 1-2 minutes by using a pipette. The aqueous mixture was then injected into a glass holder to monitor reassembly of the Au/TA-PEG membrane. The sample was then illuminated again for 5 minutes and further characterized.

Fluorescence recovery after photobleaching (FRAP)
Fluorescent membranized coacervate microdroplets were used to perform the FRAP measurements on a Leica SP5-II confocal laser scanning microscope attached to a Leica DMI 6000 inverted fluorescence microscope. A 63x oil objective lens was used for the FRAP characterization. RITC-PEG-TA was excited with a laser at 561 nm. The emission signals were collected in the range of 571-690 nm. An image was taken before photobleaching using a 561 nm laser at 100% intensity for 10 frames (1.293 s/frame). The fluorescence recovery was recorded for 10 minutes (30 s/frame). FITC-CM-dex was excited with a laser at 488 nm. The emission signals were collected in the range of 498-550 nm. An image was taken before photobleaching using a 488 nm laser at 100% intensity for 2 frames (1.293 s/frame). The florescence recovery was recorded for 1 minutes (5 s/frame). The FRAP analysis was carried out by using ImageJ software with a "Creat spectrum jru v1" plugin.

Number of PEG moles per Au nanoparticle
Typically, the caged coacervate micro-droplets were prepared using a PEG-derivative concentration of 3.6-27 μM [n(PEG)]. The concentration of Au nanoparticles was typically 3.45 x 10 13 mL -1 . The number of PEG molecules was given by n(PEG) x N A = 2.17-16.3 x 10 15 mL -1 (N A = 6.022 x 10 23 mol -1 ), indicating that on average 63 to 472 PEG molecules were attached to each Au nanoparticle.

Light-mediated uptake of ZIF8 particles in caged coacervate droplets
Microparticles of a zeolitic imidazolate framework (ZIF8) with fluorescently labelled guest bovine serum albumin molecules were prepared as follows. An aqueous solution (4.4 mL) containing 2 mg RITC-labelled BSA and 0.41 g 2-methylimidazole was mixed with aqueous Zn(NO 3 ) 2 (0.4 mL, 37 mg) under stirring at room temperature. After 30 min, the product (RITC-BSA@ZIF8) was collected by centrifuging at 4 000 rpm for 6 min and washed twice with deionized water. The prepared microparticles (mean size = 0.5 μm) were then dispersed in 5 mL deionized water.
10 μL of a freshly prepared aqueous dispersion of RITC-BSA@ZIF8 particles were gently added to a region of the freshly prepared membranized coacervate microdroplet suspension and stirred using a pipette for 10 seconds. 60 μL of the solution was then injected into a pegylated glass sample holder and left for several minutes to allow the caged coacervate droplets to settle. The droplets were illuminated for 6 min from the top of the sample holder at a distance of approximately 1 cm using light with a wavelength of 290-390 nm. CLSM images were recorded before and after light illumination.

Light-mediated capture of polyoxometalate coacervate vesicles (PCVs) by caged coacervate droplets
PCVs were prepared as follows. PDDA (12.5 mM, 400 μL, pH 6.5), ATP (2.5 mM, 400 μL, pH 6.5) and RITC-labelled amylase (2 mg/mL, 10 μL) solutions were added to a 1.75 mL vial under sonication to form PDDA/ATP coacervate microdroplets. PTA (20 mM, 100 μL) was then quickly injected, and the solution sonicated for another one minute to produce stable PCVs. The PCVs were centrifuged and the supernatant was replaced with the same volume of deionized water. The washing process was repeated two times. The washed PCVs were dispersed in 100 μL deionized water.
An aqueous mixture containing 10 μL PCV and 50 μL of Au/TA-PEG-caged coacervate droplet dispersions was injected into a pegylated glass sample holder and the microscale objects allowed to sediment for 5 minutes. The mixed dispersion was then illuminated for 10 min from the top of the sample holder at a distance of approximately 1 cm using light with a wavelength of 290-390 nm, and monitored in situ by optical microscopy.

Preparation of silica colloidosomes
An aqueous PBS buffer solution (pH=7) containing 20 mg/mL RITC-labelled amylase was dispersed into 3 mL toluene containing 35 mg of partially hydrophobic SiO 2 nanoparticles. The aqueous mixture was sonicated for 3-4 minutes to produce a water-in-oil Pickering emulsion. Immediately after emulsification, 10 μL of tetramethoxysilane was added and the liquid mixture rotated for 24 hours. The crosslinked Pickering emulsion (colloidosomes) was transferred into an aqueous solution by continuous washing with 70% ethanol : water, 50% ethanol : water followed by pure water. The colloidosomes were dispersed in 1.0 mL water.

Preparation of PEG-tagged colloidosomes
An aqueous PBS buffer solution (pH=7) containing 20 mg/mL FITC-labelled amylase was dispersed into 3 mL toluene containing 35 mg of partially hydrophobic SiO 2 nanoparticles. The aqueous mixture was sonicated for 3-4 minutes to produce a water-in-oil Pickering emulsion. Immediately after emulsification, 10 μL tetramethoxysilane was added and the liquid mixture rotated for 8 hours. 10 μL of 3-[methoxy(polyethyleneoxy)propyl]trimethoxysilane was then added and the liquid mixture rotated for 16 hours. The crosslinked Pickering emulsion (PEGtagged colloidosomes) was transferred into aqueous solution by continuous washing with 70% ethanol : water, 50% ethanol : water followed by pure water. The PEG-tagged colloidosomes were dispersed into 1.0 mL water.

Light-mediated capture of colloidosomes
10 μL of a freshly prepared aqueous dispersion of RITC-amylase-containing colloidosomes were mixed with 90 μL of an aqueous suspension of caged coacervate droplets and stirred using a pipette for 10 seconds. 60 μL of the mixture was injected into a sample holder and illuminated with light (290-390 nm) from the top of the sample holder at a distance of ca. 1cm. After 10 minutes of light illumination, the sample was stirred using a pipette for 1 min. The sample was characterized by CLSM before and after light illumination.

NMR spectroscopy studies of TA-AE-PEG hydrolysis
Hydrolysis of TA-AE-PEG as a function of pH (from pH 7.0 to pH 5.3) was investigated. 10-15 mg of TA-AE-PEG was dissolved in deuterated phosphate buffer solution (PBS, 100 mM, 0.5 mL) prepared at different pH values. The NMR spectra were acquired in a one-hour period with a 15-minute interval. The acquired spectra were analysed by integrating the peaks at 1.33 ppm (ketal) and 2.16 ppm (acetone).

Chemical-mediated capture of PCVs
Caged coacervate droplets were prepared by using Au/TA-AE-PEG6k nanoparticles as a nanoparticle surfactant. 50 μL of the aqueous dispersion was mixed with 10 μL of FITC-labelled GOx aqueous solution (final concentration, 0.2 mg/mL). The FITC-labelled GOx preferentially partitioned into the caged coacervate droplets by transport through the nanoporous membrane. The aqueous suspension of caged coacervate droplets was then mixed with 10 μL of RITC-labelled PCVs. Addition of glucose (final concentration, 10 mM) triggered the chemical-mediated capture of the PCVs. The protocells were characterized before and 60 minutes after the addition of glucose.

FACS analysis
FACS characterization was carried out using a Canto II flow cytometer operated at a low pressure with a 100 μm sorting nozzle. At least 10 6 particles were characterized to determine the 2D dot plots of the FSC and SSC light. The fluorescence signals of the individual particles were characterized by 488 and 565 nm lasers. Sample characterizations for FACS were as follows: (i) an aqueous mixture containing 20 μL of RITC-labelled PCVs and 280 μL water was characterized to determine the FACS signal for an individual population of PCVs ; (ii) 300 μL aqueous suspension containing the FITC-GOx-loaded caged coacervate droplets was characterized to determine the FACS signal of an individual population of caged coacervate micro-droplets; (iii) for chemical-mediated uncaging, an aqueous suspension containing 20 μL RITC-labelled PCVs and 300 μL GOx-loaded (0.2 mg/mL) caged coacervate droplets were characterized by FACS before and 1 hour after the addition of 10 mM glucose. The aqueous suspension was pipetted for 10 seconds before FACS characterization. Time series of counting were obtained and FlowJo 10.6 software was used for all the data analysis.

Chemical-mediated protocell sorting
An aqueous suspension containing 10 μL of a dispersion of PEG-tagged colloidosomes (FITClabelled) and 10 μL of a PCV (RITC labelled) dispersion was mixed with 100 μL GOx-loaded (0.2 mg/mL) caged coacervate droplets. 10 mM glucose was added into the aqueous suspension and the system left to react for 1 hour. The aqueous suspension was pipetted for 1 minute and then left tp settle down for 3 minutes. 50 μL aqueous suspension was gently withdrawn from the top layer of the aqueous suspension, and diluted to 300 μL by addition of deionized water for FACS characterization. An aqueous suspension containing 10 μL of a dispersion of PEG-tagged colloidosomes (FITC-labelled) and 10 μL of a PCVs (RITC labelled) dispersion was diluted by addition of 280 μL water for FACS characterization. The aqueous suspension was left to react for 1 hour after the addition of glucose. Samples were also characterized by CLSM.

Preparation of fluorescently labelled enzymes
Enzyme solutions (4 mg/mL) were prepared by dissolving the enzyme powder in 10 mL of 100 mM carbonate buffer at pH=9. 200 μL of a DMSO solution of FITC or RITC (2 mg/mL) was added dropwise and the reaction was magnetically stirred for 12 hours at 4 °C, purified by dialysis (12 hours, 2.4 L water replaced three times) and then lyophilized. The prepared fluorescently labelled enzymes were stored at -20 °C under an argon atmosphere.

Pegylation of coverslips and preparation of the sample holder
Coverslips were pegylated to reduce coacervate wetting. Firstly, the coverslips were rinsed by ethanol and dried with compressed air. Then, the coverslips were incubated at room temperature for overnight in a toluene solution containing 1 vol% 3-[methoxy(polyethyleneoxy)propyl]trimethoxysilane. Finally, the treated coverslips were rinsed with ethanol, dried with compressed air, and stored in a desiccator for further use. Sample holders used for sample observation were prepared by mounting a pegylated coverslip onto a commercial glass slide with a home-made aperture (hole diameter, 8 mm). The treated coverslip was bonded to the glass slide via UV-curing glue to seal one side of the aperture. The sample was injected into the aperture and mounted onto the treated coverslip for observation. The role of the drilled glass slide was to confine the sample solution on the treated coverslip and avoid water evaporation.

Light sources
We used a MAX-303 Xenon Light Source (300W) equipped with UV and VIS modules. 290-390 nm light was generated by the UV module without a bandpass filter. 500-520 nm light or 480-500 nm light were generated by the VIS module together with the corresponding bandpass light filter. A quartz light guide (inner diameter, 5mm; length, 1m) was connected to the light source and used for light transmission. The light intensity of the UV module was measured by an accumulated UV power meter UIT-150 S365 Uship and was 1699 mW/mm 2 . The distance between the light source and detector was 10 mm. The whole illumination area was a circle with diameter of ca. 9mm. For more information of the light source, see https://www.gmp.ch/htmlarea/pdf/asahi_pdf/max303techinfo.pdf

Movie 1.
Optical microscopy video showing light-mediated capture of PCVs in two Au/TA-PEG caged coacervate droplets present amongst a dense population of PCVs (smaller objects). Interaction with the PCVs results in translocation into the caged coacervate droplets. Movie is shown at x15 of real-time speed. Figure S1.        .5 x 10 14 particles per mL. Error bars represents the standard deviation (n=100). A minimum in the droplet diameter is observed at an optimal MTA-PEG concentration of approximately 12.5 μM. This is because at concentrations below 12.5 μM, increasing the MTA-PEG concentration generates more Janus-like nanoparticle surfactants, which enables a higher surface area to be stabilized by the surface-active properties of Au/MTA-PEG. This is similar to a conventional Pickering emulsion system. However, at concentrations above 12.5 μM, an increasing proportion of the Au nanoparticles become completely covered in the polymer, which hampers their surface activity. Thus, the diameter of the caged droplets increases and the system becomes more polydisperse. Figure S10. Self-assembly mechanism of the caged coacervate micro-droplet formation. (a) Addition of coacervate droplets into an aqueous dispersion of tannic acid-protected Au nanoparticles leads to the spontaneous accumulation of the Au nanoparticles in the coacervate droplets, indicating that the tannic acid-coated Au nanoparticles strongly interact with the coacervate droplets. Photographs show an aqueous dispersion of Au nanoparticles before (a 1 ) and after (a 2 ) the addition of the coacervate phase. The Au nanoparticles are sequestered into the coacervate phase (red arrow in (a 2 )). (b) Addition of coacervate droplets into an aqueous solution of RITC-labelled TA-PEG showing that TA-PEG is excluded from the coacervate phase and remains in the continuous aqueous phase. (b 1 ) CLSM image showing red fluorescence from RITC-PEG-TA only in the external continuous phase of a suspension of PDDA/CM-dex coacervate droplets (black circles, no fluorescence). (c) Addition of TA-PEG into a tannic acidprotected Au nanoparticle solution leads to ligand exchange on the Au nanoparticles. Because of this, addition of coacervate droplets does not result in uptake and the nanoparticles remain in the continuous phase, as confirmed by the CLSM image (c 1 ) and optical image (c 2 ). The red arrow delineates the coacervate phase. (d) Addition of tannic acid-protected Au nanoparticles to a coacervate droplet suspension followed within a few seconds by addition of RITC-labelled TA-PEG leads to spontaneously membranization of the coacervate droplets, implying that interfacial assembly occurs due to an asymmetric ligand coverage associated with the in situ formation of amphiphilic nanoparticles. (e) Addition of tannic acid-protected Au nanoparticles to a coacervate droplet suspension followed by stirring for 5 minutes leads to the accumulation of the Au nanoparticles in the coacervate droplets. If RITC-labelled TA-PEG is then added to the continuous phase along with vigorous stirring, membranization of the coacervate droplets occurs, consistent with an asymmetric distribution of ligands. (e 1 ) CLSM image shows the membranized coacervate droplets. Scale bars are 50 μm.   ME nanoparticle-caged coacervate microdroplets recorded before (1), 30s after bleaching (2), 5 minutes after recovery (3) and 10 minutes after recovery (4). The photobleached area is delineated by white rectangles and corresponds to the jammed nanoparticle membrane. Red fluorescence, RITC-labelled TA-PEG. As observed for bidentate Au/TA-PEG, a solid-like membrane is also produced using the monodentate ME ligand. Scale bar is 20 μm. (b) Plots of changes in fluorescence intensity for delineated area shown in (a). Error bars represent the standard deviation (n=3).       Figure S20. Overlays of bright field and fluorescence CLSM images of an aqueous dispersion of caged Au/TA-PEG coacervate droplets and BSA@ZIF8 microparticles before (a) and after 6 minutes of light exposure (b) showing triggered uptake into the coacervate interior (red fluorescence); corresponding changes in mean fluorescence recorded inside and outside the caged coacervate droplets, as well as without light illumination (normal daylight, control) are shown in (c). Error bars in (c) represent the standard deviation (n = 10). Scale bars, 50 μm.